Gene Targeting in a Novel Mouse Model and the Chicken DT40 Cell Line

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Gene targeting has the power to create precise changes at specific sites within the genome. In the context of gene therapy, this technology may be used to treat patients with monogenic diseases by fixing mutations in disease causing genes, followed by transplanting the corrected cells back into the patient. However, the natural rate of gene targeting is too low to be of practical use in most cells; an exception to this is the chicken DT40 cell line which has a high relative rate of gene targeting (gene targeting rate/ random integration rate). We therefore sought to determine the basis of this high rate of gene targeting using assays which quantitate the rates of repairing DNA double-strand breaks through different repair pathways. We show that compared to other cell types, DT40 cells are deficient in random integration. Furthermore, we show this deficiency is due to a reduced ability to repair DNA breaks lacking homology at the ends. In other cell types, the naturally low rate of gene targeting can be stimulated 30-40,000 fold by inducing a double-strand break at the target site. These breaks can be created by proteins called zinc finger nucleases (ZFNs). ZFN mediated gene targeting is a powerful technology, but has not yet been fully characterized in primary cells. Furthermore, before clinical use in the treatment of monogenic diseases, it is necessary to first test this technology in animal models. In the second portion of this dissertation, we developed a mouse model of a generic recessive genetic disease. This model allows the study of gene targeting in any cell population isolated from the mouse. Using this model, we demonstrate ZFN mediated gene targeting in variety of primary cells isolated from the mouse, including ES cells, fibroblasts, and astrocytes. We further demonstrate that targeted stem cells retain their pluripotency, and show that targeted fibroblasts can be transplanted back into a recipient and continue to express protein from the corrected gene. This body of work contributes to bringing the technology of gene targeting closer to clinical application by detailing methods which can be used to further increase gene targeting rates, as well as providing a paradigm in which to study gene targeting followed by transplantation